| Abstract | merička gnjiloća medonosne pčele (AGMP) je zarazna bolest nepoklopljenog i
poklopljenog pčelinjeg legla koja pčelarstvu nanosi višestruke štete. Uzročnik bolesti je
bakterija Paenibacillus larvae koja u nepovoljnim životnim uvjetima tvori dugo živuće i
otporne spore. Patogeneza, klinička slika i stupanj virulentnosti kod AGMP ovise o genotipu
bakterije P. larvae. Dosad je utvrđeno pet genotipova P. larvae (ERIC I, ERIC II, ERIC III,
ERIC IV, ERIC V) koji se međusobno razlikuju u morfologiji, biokemijskim čimbenicima,
virulenciji te čimbenicima koji utječu na virulenciju. Poznavanje raširenosti i dinamike
pojavnosti pojedinih genotipova P. larvae na određenom području pruža uvid u patofiziološke
procese na razini ličinke / pčelinje zajednice te utječe na procjenu rizika od AGMP jer postoji
značajna korelacija između genotipa i pojavnosti vidljivih kliničkih znakova. Tvrdokoran tijek
bolesti, otpornost uzročnika, poteškoće u kliničkoj dijagnostici i suzbijanju učinili su AGMP
jednom od najtežih u svijetu. Bolesne pčelinje zajednice bez provedbe posebnih mjera ne mogu
ozdraviti, a primjena antibiotika u liječenju bolesti nije dozvoljena zbog moguće pojave rezidua
u pčelinjim proizvodima, rezistencije uzročnika te spoznaje da antibiotici djeluju samo na
vegetativne oblike bakterije P. larvae, a ne uništavaju spore što doprinosi horizontalnom širenju
bolesti u pčelinjacima. Ponekad je bolesnu pčelinju zajednicu najbolje sanirati spaljivanjem
zajedno s košnicom i onečišćenim priborom. Nakon provedenih sanacijskih mjera nužno je
primjeniti učinkovitu završnu dezinfekciju opreme, pribora i pčelinjaka čiji uspjeh ovisi o
izboru dezinficijensa, preporučenoj koncentraciji radnih otopina, načinu i dužini trajanja
aplikacije, vrsti mikroorganizama koji se moraju ukloniti te površini/materijalu koji se
dezinficira. Cilj ovog istraživanja bila je provedba genotipizacije bakterije P. larvae na pedeset
terenskih izolata izdvojenih iz karakteristično promijenjenih uginulih pčelinjih ličinki
skupljenih u razdoblju od jedanaest godina (2010. – 2020.), te tako utvrditi raširenost i
učestalost određenih genotipova u Republici Hrvatskoj (RH). Također, cilj je bio i utvrđivanje
učinka deset komercijalno dostupnih i uobičajeno korištenih dezinficijensa u pčelarstvu, na
terenske i certificirane sojeve bakterije P. larvae, u laboratorijskim uvjetima te uspoređivanje
dobivenih rezultata među pojedinim genotipovima bakterije P. larvae. Genotip ERIC I bakterije
P. larvae utvrđen je u pčelinjim zajednicama na području RH u visokoj prevalenciji od 90,3 %,
a genotip ERIC II u niskoj prevalenciji od 7,3 % od ukupno uspješno analiziranih terenskihXI
izolata. Za jedan izolat postavljena je sumnja na genotip ERIC IV s prevalencijom od 2,4 % te
je potrebna daljnja verifikacija takvog nalaza. Istraživani su učinci Genoxa, Genolla s pjenom,
Ecocid S, Sekusept aktiv, Incidin OxyFoam S, Bee Protect H forte, Bee Protect F, Despadac,
Despadac Secure i EM® PROBIOTIK ZA PČELE u testu stvaranja zone inhibicije,
suspenzijskom testu učinka na vijabilne bakterije P. larvae, testu dezinficijskog djelovanja na
površinama i suspenzijskom testu učinka na spore bakterije P. larvae. Učinak dezinficijensa
Genoxa na bakteriju P.larvae nije pokazao poželjan sporocidni profil zbog predugog vremena
koje je potrebno da bi se ostvario sporocidni učinak te je isti limitiran dok proizvod Genoll s
pjenom nije uopće pokazao sporocidno djelovanje. Proizvodi iz linije Despadac u
suspenzijskom testu i u testu na površinama pokazali su baktericidno djelovanje, ali sporocidni
učinak nije zadovoljavajući zbog slabijeg učinka u kontaktnom vremenu od 30 minuta.
Suspenzijskim testom nije utvrđen zadovoljavajući sporocidni učinak Bee protect proizvoda, a
proizvod EM® PROBIOTIK ZA PČELE nije pokazao u testu agar gel difuzije značajan
baktericidni učinak. Sekusept aktiv u 2% koncentraciji i Incidin OxyFoam S u suspenzijskom
testu sporocidnog djelovanja pokazali su zadovoljavajući sporocidni učinak na sva četiri
genotipa bakterije P. larvae (ERIC I do ERIC IV). |
| Abstract (english) | INTRODUCTION
American foulbrood (AFB) is a contagious disease of sealed and unsealed honeybee
brood that causes multiple damage to beekeeping. The causative agent of the disease is the
bacterium Paenibacillus larvae, which in unfavorable life conditions forms long-lived and
resistant spores. The infectious forms of P. larvae are spores, and susceptible to infection are
honeybee larvae at the age when they are taking food. It takes only ten spores to infect one
honeybee larvae younger than one day, but with the time passing by, susceptibility decreases
and more than ten million spores are needed to infect a larva between four and five days old.
Moreover, the number of spores required to cause infection in the later stages of honeybee
larvae development is so high that a naturally infection is not possible. One dead larva can
contain up to 2.5 billion newly created infectious spores. The pathogenesis, clinical signs, and
degree of virulence in AFB depend on the P. larvae genotype. Recently, five genotypes of P.
larvae (ERIC I, ERIC II, ERIC III, ERIC IV, ERIC V) have been identified, which differ in
morphology, biochemical parameters, virulence, and factors influencing virulence. The
virulence of P. larvae is conditioned by the possibility of infecting the honeybee larva and the
time required until the death of the infected larva. The formation of a large amount of longlived and resistant spores, together with the possibility of multiplication and development of
vegetative forms of the bacterium allows a high probability of P. larvae infection. The analysis
of pathogenicity or virulence showed significant differences between genotypes ERIC I to
ERIC V. The ERIC I genotype of P. larvae takes 12 days to cause death of all infected larvae
(LT100 = 12 days), while the genotypes ERIC II to ERIC IV take only seven days. Therefore,
according to the rate of larval death, these P. larvae genotypes are classified into three groups:
ERIC I - slow leads to death of infected honeybee larvae, ERIC II - moderately fast leads to
death of infected larvae, and ERIC III to ERIC V - are genotypes of pathogens that quickly lead
to the death of the infected larva. The LT100 results for genotype ERIC II indicated that all
infected larvae would die before the brood cells could be sealed. In this way, the adult honeybee
workers have enough time to perform their hygienic skills, removing the dead larvae from brood
cells. At the same time, the process of creating spores at the level of the honey bee colony would
be disrupted, contributing to slow down the spread and development of the disease. However,
the ERIC I genotype is less virulent at the level of a single larva because infected larvae die
after sealing the cells. Consequently, the removal of dead larvae is reduced in such cases, and
the possibility of producing and spreading the causative spores is significantly increased.
Ultimately, genotype ERIC I is highly virulent for the honeybee colony leading to its rapid
decline when compared to ERIC II which shows lower virulence at the honeybee colony level
and slower decline of honeybee colony thereby showing a negative correlation of P. larvae
virulence at the larval level, and consequently at the honeybee colony level. The knowledge of
the distribution and dynamics of occurrence of individual P. larvae genotypes in a given area
provides insight into pathophysiological processes at the level of the larva or honeybee colony
and influences the risk assessment of AFB once there is a significant correlation between
genotype and clinical signs. Vegetative rods of P. larvae have long, peritrichous arranged cilia
that allow active movement in the form of a swarm motility. It was found that the P. larvae
ERIC II genotype can move superficially in the form of a swarm and form a free-floating
biofilm, while the P. larvae ERIC I genotype can form a biofilm but cannot move in the form
of a swarm. These facts, that P. larvae is able to produce biofilm and actively move in the form
of a swarm, requires new approaches in diagnostic and disenfection measures. The persistent
course of the disease, resistance of pathogens, difficulties in clinical diagnosis and control, have
made AFB one of the most difficult honeybee disease in the world. Infected honeybee colonies
can hardly recover without implementation of special measures. The use of antibiotics in the
treatment of diseases is prohibited due to the appearance of residues in honeybee products,
resistance of the pathogen and the knowledge that antibiotics act only on vegetative forms of
P. larvae, thereby providing horizontal spread of disease in apiaries. Most of the times, the best
way to sanitate an infected honeybee colony it’s by burning it together with the hive and hive
tools.
During and after the implementation of eradication measures, it is necessary to carry out
final disinfection, which success depends on the choice of effective disinfectant, recommended
concentration of solutions, method and duration of application, type of microorganisms to be
removed, surfaces and material to be disinfected. From the epizootiological point of view,
disinfection can be preventive or focal. Preventive disinfection includes procedures and
measures when infectious disease is not present in the apiary or its immediate surroundings. It
is regularly carried out within the guidelines of good beekeeping practice and is an integral part
of normal hygiene in the apiary. Keeping beehives, equipment, hive tools, food and water for
bees clean is the basis of preventive disinfection. Focal disinfection is carried out when an
infectious disease is present in the apiary and aims to remove microorganisms from the foci of
infection, thus preventing its further spread. Depending on the method of execution, it can be
continuous and final. Continuous disinfection involves systematic and repetitive procedures
from the moment of the outbreak of infection in the apiary. It can be combined with veterinary
administrative measures to cure diseases, such as burning bee colonies. The final disinfection
has been considered as a one-step procedure after the implemented measures of disease
remediation. Disinfection is often carried out by mechanical, physical and chemical procedures.
Mechanical processes, such as cleaning, scraping, and washing, remove impurities and organic
matter in which microorganisms are incorporated, thus facilitating the disinfection process. It
has been observed that many disinfectants are ineffective in the presence of impurities and
organic matter. Cleaning agents - detergents, soaps and abrasive powders - reduce the number
of microorganisms, removing them from surfaces and objects. Physical disinfection procedures
involve the use of moist or dry heat and radiation, where moist heat acts faster in a shorter
period of time and is more efficient compared to dry heat. Burning hives, honeybee colonies
and other accessories equipment and tools is an effective way of sanitation of AFB. Although
the scorching process completely destroys the P. larvae spores on the surface of wooden hives,
a significant number of infectious spores still remain active in internal wood structures. The
wood fibers behave like organic matter which, already in a concentration of 2 %, significantly
reduces the effect of surface disinfection. Boiling in water under normal pressure for thirty
minutes with the addition of 1 to 2 % sodium carbonate or boiling in water under pressure for
twenty minutes successfully destroys P. larvae spores in the internal wood structures. Chemical
disinfection processes include the use of various disinfectants whose choice depends on the
spectrum of the microorganism to be destroyed, the presence of organic matter on the surface,
environmental conditions, and the toxicity of the disinfectant to humans, animals, and the
environment. The aldehydes, halogen compounds and oxidants show an effect on bacterial
endospores while inorganic acids, alkaline salts and phenols have a limited effect. High and
rapid sporocidal activity of glutaraldehyde, sodium hypochlorite and caustic soda on P. larvae
spores was found. The aim of this study was to implement genotyping of P. larvae on fifty field
samples isolated from characteristically altered dead honeybee larvae over a period of eleven
years (2010-2020), and thus determine the prevalence and frequency of certain genotypes in
the Republic of Croatia. The effect of ten commercially available and commonly used
conditions was also determined. The ultimate goal was to perform the comparison of the results
obtained on the effect of tested disinfectants with individual genotypes of P. larvae.
MATERIAL AND METHODS
The P. larvae bacteria used in the study came from two sources. One was a validated
strain (German Collection of Microorganism and Cell Culture - DSMZ; Braunschweig,
Germany) of which four genotypes were used (DSM 7030 (ERIC I), DSM 25430a (ERIC II),
LMG 16252 (ERIC III) and LMG 16247 (ERIC IV)). For an additional verification of the
obtained results, genotyped strains collected in the Republic of Croatia for several years were
used. The P. larvae strains were cultured on Columbia sheep blood agar (BD), and for liquid
culture, P. larvae strains were grown in brain heart infusion medium (BHIM).
For the genotyping study, the primers ERIC1R and ERIC2 were used, and the method
of repetitive extragenic paliandromic sequence-PCR (REP-PCR) was performed to determine
which genotype our samples belonged to. The isolation of genomic DNA was performed with
a commercial QIAamp Mini DNA blood and tissue kit according to the manufacturer's
instructions, with special preparation of bacteria for isolation.
The commercially disinfectants were selected based on the recommendations of
producers and beekeepers, as well as their availability on the market. The following
disinfectants were used: Bee Protect products (Bee Protect H forte and Bee Protect F), Genox,
Genoll with foam, Despadac, Despadac Secure, Ecocid S, Sekusep aktiv, Incidin Oxyfoam S
and EM® probiotic for bees. Selected products were tested by 1) determining the zone of
inhibition in agar diffusion test, 2) suspension test for viable bacteria, 3) surface disinfectant
test, and 4) sporocidal effect in suspension test for all four genotypes of P. larvae (ERIC I to
ERIC IV).
RESULTS
The research and sampling on apiaries in the Republic of Croatia during the period from
2010 to 2020, of the total successfully analyzed P. larvae samples (n = 41), most belonged to
the ERIC I genotype (90.3 %), while only three samples belonged to genotype ERIC II (7.3 %).
Nine samples were not suitable for interpretation. The finding of one sample suspected to be of
ERIC IV genotype (2.4%) would need further verification, especially from the point of view
that ERIC III and ERIC IV genotypes have not been isolated from field samples for decades
and are present only in archived collections of bacterial cultures. Since there is no anamnestic
data, it can be assumed that the beekeeper used old equipment, wax, honey and / or food
additives of unknown origin. The extremely high proportion of isolates belonging to the ERIC I genotype can be explained by the lack of systematic monitoring on AFB such as regular
clinical examinations before moving bees on honey pasture, or early diagnosis thereby
examining honey, adult bees or hive debris from the botom board for P. larvae spores.
Moreover, the subject samples were taken from honeybee colonies where beekeepers and
veterinarians had already raised the suspicion of AFB based on typical clinical signs. This
correlates with the comprehension that the ERIC I genotype is less virulent at the level of a
single larva, so infected honeybee larvae die after sealing cells and clinical signs of disease can
be clearly seen because workers have not removed the dead larvae. Therefore, it is a logical
recommendation to develop a new model for monitoring honeybee colonies on AFB in the
Republic of Croatia, which would include active and passive monitoring and early diagnosis of
AFB as an act of determining honeybee colonies germ carriers or reservoirs of disease, as well
as prevention of disease in apiaries.
The suspension test showed a significant effect of undiluted Genox after 15 minutes of
application depending on the duration of exposure, while in the test of disinfectant the effect on
surfaces was seen after 30 minutes and was not dependent on further prolongation of contact
time. Genoll with foam didn't show sporocidal effect while Genox disinfectant at a
concentration of 10% reduced the number of spores by 1 logarithm, reaching a reducting of
three logarithms in 30 and 60 minutes when 100% concentration was used. The observed effect
of Genox disinfectant on P. larvae didn't show a desirable sporocidal profile due to the too long
time required to achieve a sporocidal effect. In the suspension test and in the surface test, Incidin
OxyFoam S showed antimicrobial activity on vegetative forms of P. larvae after only one
minute, and sporocidal action at the level of reduction of 6 logarithms after 30 minutes of
contact time. The tested effect of the disinfectant Incidin OxyFoam S on P. larvae showed a
satisfactory sporocidal effect on all four genotypes of P. larvae (ERIC I to ERIC IV). The
products from the Despadac line showed bactericidal activity in the suspension test and in the
surface test, but the sporocidal effect was not satisfactory due to the poorer effect in the contact
time of 30 minutes. The bactericidal effect of Ecocid S and Sekusept aktiv on P. larvae was
determined by agar gel diffusion test and suspension test. The ATP bioluminescence test in the
surface test of Ecocid S and Sekusept aktiv showed a bactericidal effect whereas Sekusept aktiv
showed a desirable sporocidal effect in the suspension test at a concentration of 2% on all four
genotypes of P. larvae (ERIC I to ERIC IV). The Bee protect products showed in the agar
diffusion test a bactericidal effect on P. larvae. In the suspension test for viable bacteria, the
bactericidal effect for Bee protect H forte was determined, while in the test of action on surfaces,
Bee protect products didn't show any bactericidal effect on P. larvae. The suspension test didn'tXVII
show any sporocidal effect of Bee protect products. The EM probiotic product for bees didn't
show significant bactericidal effect in the agar gel diffusion test.
DISCUSSION
In our research on apiaries in the Republic of Croatia in the period from 2010 to 2020,
most field isolates belonged to the ERIC I genotype (90.3%), while only three isolates belonged
to the ERIC II genotype (7.3%). In one isolate of P. larvae, the ERIC IV genotype (2.4%) was
suspected and further verification of such a finding is required. The extremely high proportion
of P. larvae isolates belonging to the ERIC I genotype can be explained by the lack of
systematic monitoring for the presence of AFB in apiaries.
Sodium hypochlorite (NaOCl) is a frequently used disinfectant and its effectiveness
depends on the concentration of available chlorine and the pH of the solution. Genox and Genoll
show an inhibitory effect on vegetative forms of P. larvae, but the effect of Genox after 60
minutes is slightly attenuated, which may be related to the fact that all biocides that have
chlorine as an active component have time-limited effects due to its consumption during
exposure to environmental factors. Hydrogen peroxide leads to the oxidation of lipids and
proteins of the outer layer of the bacterial cell. The sporocidal action of Incidin OxyFoam S
was determined at the level of reduction by six logarithms after 30 minutes of contact. Previous
studies performed with hydrogen peroxide did not include a combination with other substances,
or even auxiliary substances, which results in differences from our study. Quaternary
ammonium salts have been in use for many years in disinfection. Products from the Despadac
line (Despadac and Despadac Secure) showed bactericidal activity during exposure for 30
minutes, after which there was a reduction in the number of germinating spores of P. larvae,
but only by one or two logarithms. Such a result in disinfection conditions is not satisfactory,
but the use of Despadac products in sanitation can be considered. Peracetic acid is considered
a potent biocide, even at low concentrations in the presence of organic residues, and is degraded
to non-toxic substances. The action of peracetic acid as a disinfectant is not dependent on
ambient temperature. Studies have shown that the effectiveness of peracetic acid varies
depending on whether the microorganisms are in suspension or on the surface, which was not
the case in our study during the study Sekusept Aktiv and Ecocida S. The Bee protect products
caused a certain effect that increased with the time of exposure of bacteria to disinfectant.
However, this effect did not meet the set standards and Bee protect products can't be
recommended for use in the final disinfection of equipment, utensils and apiaries after
remediation of clinically visible AFB. The effective microorganisms are used in agriculture,XVIII
forestry, livestock, aquaculture, beekeeping, environmental protection and medicine. The
inhibitory effect of the bee food supplement EM® PROBIOTIC FOR BEES on reducing the
number of P. larvae bacteria was minimal and therefore this product can't be considered a
classic disinfectant or biocide in the true sense of the word. However, the action of effective
microorganisms in a living organism has been proven many times.
CONCLUSION
The effective application of control measures and proper application of final disinfection
can reduce the appearance of a visible clinical signs of AFB whereas methods of early diagnosis
can significantly reduce the incidence of the disease. |
